1. Drug Distribution and Protein Binding Course Title : Biopharmaceutics and Pharmacokinetics – I

2. Physiologic Factors of Distribution After a drug is absorbed into plasma, the drug molecules are distributed throughout the body by the systemic circulation. The drug molecules are carried by the blood to the target site (receptor) for drug action and to other (non-receptor) tissues as well, where side effects or adverse reactions may occur. Drug molecules are distributed to eliminating organs, such as the liver and kidney, and to non-eliminating tissues, such as the brain, skin, and muscle. In pregnancy, drugs cross the placenta and may affect the developing fetus.

3. Physiologic Factors of Distribution Drugs can also be secreted in milk via the mammillary glands. A substantial portion of the drug may be bound to proteins in the plasma and/or tissues. Lipophilic drugs deposit in fat, from which the drug may be slowly released. The circulatory system consists of a series of blood vessels; these include the arteries that carry blood to tissues, and the veins that return the blood back to the heart. An average subject (70 kg) has about 5 L of blood, which is equivalent to 3 L of plasma.

4. Physiologic Factors of Distribution About 50% of the blood is in the large veins or venous sinuses. The volume of blood pumped by the heart per minute—the cardiac output—is the product of the stroke volume of the heart and the number of heart beats per minute. An average cardiac output is 0.08 L/left ventricle contraction x 69 contractions (heart beats)/min, or about 5.5 L/min in subjects at rest. The cardiac output may be five to six times higher during exercise.

5. Physiologic Factors of Distribution Left ventricular contraction may produce a systolic blood pressure of 120 mm Hg, and moves blood at a linear speed of 300 mm/sec through the aorta. Mixing of a drug solution in the blood occurs rapidly at this flow rate. Drug molecules rapidly diffuse through a network of fine capillaries to the tissue spaces filled with interstitial fluid. The interstitial fluid plus the plasma water is termed extracellular water, because these fluids reside outside the cells. Drug molecules may further diffuse from the interstitial fluid across the cell membrane into the cell cytoplasm.

6. Major water volumes (L) in average 70–kg human.

7. Diffusion of drug from capillaries to interstitial spaces.

8. Physiologic Factors of Distribution Drug distribution is generally rapid, and most small drug molecules permeate capillary membranes easily. The passage of drug molecules across a cell membrane depends on the physicochemical nature of both the drug and the cell membrane. Cell membranes are composed of protein and a bilayer of phospholipid, which act as a lipid barrier to drug uptake.

9. Physiologic Factors of Distribution Thus, lipid-soluble drugs generally diffuse across cell membranes more easily than highly polar or water-soluble drugs. Small drug molecules generally diffuse more rapidly across cell membranes than large drug molecules. If the drug is bound to a plasma protein such as albumin, the drug–protein complex becomes too large for easy diffusion across the cell or even capillary membranes.

10. Diffusion and Hydrostatic Pressure The processes by which drugs transverse capillary membranes include passive diffusion and hydrostatic pressure. Passive diffusion is the main process by which most drugs cross cell membranes. Passive diffusion is the process by which drug molecules move from an area of high concentration to an area of low concentration.

11. Diffusion and Hydrostatic Pressure Passive diffusion is described by Fick's law of diffusion: Rate of drug diffusion = dQ = - DKA (C P – C t ) dt h where C p – C t is the difference between the drug concentration in the plasma (C p ) and in the tissue (C t ), respectively; A is the surface area of the membrane; h is the thickness of the membrane; K is the lipid–water partition coefficient; and D is the diffusion constant. The negative sign denotes net transfer of drug from inside the capillary lumen into the tissue and extracellular spaces.

12. Diffusion and Hydrostatic Pressure Diffusion is spontaneous and temperature dependent. Diffusion is distinguished from blood flow-initiated mixing, which involves hydrostatic pressure. Hydrostatic pressure represents the pressure gradient between the arterial end of the capillaries entering the tissue and the venous capillaries leaving the tissue. Hydrostatic pressure is responsible for penetration of water-soluble drugs into spaces between endothelial cells and possibly into lymph.

13. Diffusion and Hydrostatic Pressure In the kidneys, high arterial pressure creates a filtration pressure that allows small drug molecules to be filtered in the glomerulus of the renal nephron. Blood flow-induced drug distribution is rapid and efficient, but requires pressure. As blood pressure gradually decreases when arteries branch into the small arterioles, the speed of flow slows and diffusion into the interstitial space becomes diffusion or concentration driven and facilitated by the large surface area of the capillary network.

14. Diffusion and Hydrostatic Pressure The average pressure of the blood capillary is higher (+18 mm Hg) than the mean tissue pressure (–6 mm Hg), resulting in a net total pressure of 24 mm Hg higher in the capillary over the tissue. This pressure difference is offset by an average osmotic pressure in the blood of 24 mm Hg, pulling the plasma fluid back into the capillary. Thus, on average, the pressures in the tissue and most parts of the capillary are equal, with no net flow of water.

15. Diffusion and Hydrostatic Pressure At the arterial end, as the blood newly enters the capillary, however, the pressure of the capillary blood is slightly higher (about 8 mm Hg) than that of the tissue, causing fluid to leave the capillary and enter the tissues. This pressure is called hydrostatic or filtration pressure. This filtered fluid (filtrate) is later returned to the venous capillary due to a lower venous pressure of about the same magnitude. The lower pressure of the venous blood compared with the tissue fluid is termed absorptive pressure. A small amount of fluid returns to the circulation through the lymphatic system.

16. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Because the process of drug transfer from the capillary into the tissue fluid is mainly diffusional, the membrane thickness, diffusion coefficient of the drug, and concentration gradient across the capillary membrane are important factors in determining the rate of drug diffusion. Kinetically, if a drug diffuses rapidly across the membrane in such a way that blood flow is the rate-limiting step in the distribution of drug, then the process is perfusion or flow limited.

17. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs A person with congestive heart failure has decreased cardiac output, resulting in impaired blood flow, which may reduce renal clearance through reduced filtration pressure and blood flow. In contrast, if drug distribution is limited by the slow diffusion of drug across the membrane in the tissue, then the process is termed diffusion or permeability limited. Drugs that are permeability limited may have an increased distribution volume in disease conditions that cause inflammation and increased capillary membrane permeability.

18. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs The delicate osmotic pressure balance may be altered due to albumin and/or blood loss or due to changes in electrolyte levels in renal and hepatic disease, resulting in net flow of plasma water into the interstitial space (edema). This change in fluid distribution may partially explain the increased extravascular drug distribution during some disease states.

19. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs

20. Blood flow, tissue size, and tissue storage are also important in determining the time it takes the drug to become fully distributed. Drug affinity for a tissue or organ refers to the partitioning and accumulation of the drug in the tissue. Distribution half life: Time for 50% drug distribution and the time for drug distribution is generally measured by the distribution half- life.

21. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs The factors that determine the distribution constant of a drug into an organ are related to the blood flow to the organ, the volume of the organ, and the partitioning of the drug into the organ tissue, as shown below : K d = Q/VR where k d = first-order distribution constant, Q = blood flow to the organ, V = volume of the organ, R = ratio of drug concentration in the organ tissue to drug concentration in the blood (venous).

22. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs The distribution half-life of the drug to the tissue, t d1/2 may easily be determined from the distribution constant, t d1/2 = 0.693/k d. The ratio R must be determined experimentally from tissue samples. Pharmacokineticists have estimated the ratio R based on knowledge of the partition coefficient of the drug.

23. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs The partition coefficient (P o/w ) is defined as a ratio of the drug concentration in the oil phase (usually represented by octanol) divided by the drug concentration in the aqueous phase measured at equilibrium under specified temperature in vitro in an oil/water two-layer system. The partition coefficient is one of the most important factors that determine the tissue distribution of a drug.

24. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs If each tissue has the same ability to store the drug, then the distribution half-life is governed by the blood flow, Q, and volume (size), V, of the organ. A large blood flow, Q, to the organ decreases the distribution time, whereas a large organ size or volume, V, increases the distribution time because a longer time is needed to fill a large organ volume with drug. Fig. 10-5 illustrates the distribution time (for 0, 50, 90, and 95% distribution) for the adrenal gland, kidney, muscle (basal), skin, and fat tissue in an average human subject (ideal body weight, IBW = 70 kg).

25. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Vascular tissues such as the kidneys and adrenal glands achieve 95% distribution in less than 2 minutes. In contrast, drug distribution time in fat tissues takes 4 hours, while less vascular tissues, such as the skin and muscles, take between 2 and 4 hours (Fig. 10-5). When drug partition of the tissues is the same, the distribution time is dependent only on the tissue volume and its blood flow.

26. Drug distribution in five groups of tissues at various rate of equilibration. (1 = adrenal, 2 = kidney, 3 = skin, 4 = muscle [basal], 5 = fat.)

27. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Blood flow is an important consideration in determining how rapid and how much drug reaches the receptor site. Under normal conditions, limited blood flow reaches the muscles. During exercise, the increase in blood flow may change the fraction of drug reaching the muscle tissues. Diabetic patients receiving intramuscular injection of insulin may experience the effects of changing onset of drug action during exercise.

28. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Normally, the blood reserve of the body stays mostly in the large veins and sinuses in the abdomen. During injury or when blood is lost, constriction of the large veins redirect more blood to needed areas and, therefore, affect drug distribution.

29. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Fig. 10-6 illustrates the distribution of a drug to three different tissues when the partition of the drug for each tissue varies. For example, the adrenal glands have five times the concentration of the plasma (R = 5), while for drug partition for the kidney, R = 3, and for basal muscle, R = 1. In this illustration, the adrenal gland and kidney take 5 and 3 times as long to be equilibrated with drug.

30. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Thus, it can be seen that, even for vascular tissues, high drug partition can take much more time for the tissue to become fully equilibrated. In the example in Fig 10-6, drug administration is continuous (as in IV infusion), since tissue drug levels remain constant after equilibrium.

31. Fig.10-6 Drug distribution in three groups of tissues with various ability to store drug (R) (top = adrenal; middle = kidney; bottom = muscle).

32. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs Some tissues have great ability to store and accumulate drug, as shown by large R values. For example, the antiandrogen drug flutamide and its active metabolite are highly concentrated in the prostate. The prostate drug concentration is 20 times that of the plasma drug concentration; thus, the antiandrogen effect of the drug is not fully achieved until distribution to this receptor site is complete.

33. Distribution Half-Life, Blood Flow, and Drug Uptake by Organs If a tissue has a long distribution half-life, a long time is needed for the drug to leave the tissue when blood level decreases. Understanding drug distribution is important because the activities of many drugs are not well correlated with plasma drug level. Kinetically, both protein binding or favorable solubility in the tissue site lead to longer distribution times.

34. Drug Accumulation The deposition or uptake of the drug into the tissue is generally controlled by the diffusional barrier of the capillary membrane and other cell membranes. For example, the brain is well perfused with blood, but many drugs with good aqueous solubility have high kidney, liver, and lung concentrations and yet little or negligible brain drug concentration. The brain capillaries are surrounded by a layer of tightly joined glial cells that act as a lipid barrier to impede the diffusion of polar or highly ionized drugs.

35. Is this Perfusion limited or diffusion limited? Explain.

36. Drug Accumulation Tissues receiving high blood flow equilibrate quickly with the drug in the plasma. However, at steady state, the drug may or may not accumulate (concentrate) within the tissue. The accumulation of drug into tissues is dependent on both the blood flow and the affinity of the drug for the tissue. Drug uptake into a tissue is generally reversible. The drug concentration in a tissue with low capacity equilibrates rapidly with the plasma drug concentration and then declines rapidly as the drug is eliminated from the body.

37. Drug Accumulation In contrast, drugs with high tissue affinity tend to accumulate or concentrate in the tissue. Drugs with a high lipid/water partition coefficient are very fat soluble and tend to accumulate in lipid or adipose (fat) tissue. This process is reversible, but the extraction of drug out of the tissue is so slow that the drug may remain for days or even longer in adipose tissues, long after the drug is depleted from the blood.

38. Drug Accumulation Because the adipose tissue is poorly perfused with blood, drug accumulation is slow. However, once the drug is concentrated in fat tissue, drug removal from fat may also be slow. For example, the chlorinated hydrocarbon such as DDT (dichlorodiphenyltrichloroethane) is highly lipid soluble and remains in fat tissue for years.

39. Drug Accumulation Drugs may accumulate in tissues by other processes. For example, drugs may accumulate by binding to proteins or other macromolecules in a tissue. Digoxin is highly bound to proteins in cardiac tissue, leading in a large volume of distribution (440 L/70 kg) and long elimination t 1/2 (approximately 40 hours).

40. Drug Accumulation Some tissues have enzyme systems that actively transport natural biochemical substances into the tissues. For example, various adrenergic tissues have a specific uptake system for catecholamines, such as norepinephrine. Thus, amphetamine, which has a phenylethylamine structure similar to norepinephrine, is actively transported into adrenergic tissue.

41. Drug Accumulation In a few cases, the drug is irreversibly bound into a particular tissue. Irreversible binding of drug may occur when the drug or a reactive intermediate metabolite becomes covalently bound to a macromolecule within the cell, such as to a tissue protein. Many purine and pyrimidine drugs used in cancer chemotherapy are incorporated into nucleic acids, causing destruction of the cell.

42. Apparent Volume Distribution The concentration of drug in the plasma or tissues depends on the amount of drug systemically absorbed and the volume in which the drug is distributed. The apparent volume of distribution, V D in a model, is used to estimate the extent of drug distribution in the body. Although the apparent volume of distribution does not represent a true anatomical, or physical volume, the V D represents the result of dynamic drug distribution between the plasma and the tissues and accounts for the mass balance of the drug in the body.

43. Apparent Volume Distribution Volume = amount (mg) of drug added to system (L) drug concentration (mg/ml) in system after equilibrium The above Equation describes the relationship of concentration, volume, and mass, as shown in the following equation. concentration (mg/ml) X Volume (L) = mass (mg)

44. Beaker Exercise

45. Apparent Volume Distribution The following considerations must be taken into account : 1.Drug must be at equilibrium in the system before any drug concentration is measured. In non-equilibrium conditions, the sample removed from the system for drug assay does not represent all parts of the system. 2.Drug binding distorts the true physical volume of distribution when all components in the system are not properly sampled and assayed. Extravascular drug binding increases the apparent V D.

46. Apparent Volume Distribution 3. Both intravascular and extravascular drug binding must be determined to calculate meaningful volumes of distribution. 4. The apparent V D is essentially a measure of the relative extent of drug distribution outside the plasma compartment. Greater tissue drug binding and drug accumulation increases V D, whereas greater plasma protein drug binding decreases the V D distribution. 5. Undetected cellular drug metabolism increases V D.

47. Apparent Volume Distribution 6.An apparent V D larger than the combined volume of plasma and body water is indicative of (4) and (5), or both, above. 7.Although the V D is not a true physiologic volume, the V D is useful to relate the plasma drug concentration to the amount of drug in the body. This relationship of the product of the drug concentration and volume to equal the total mass of drug is important in pharmacokinetics.

48. Apparent Volume Distribution The apparent volume of distribution, in general relates the plasma drug concentration to the amount of drug present in the body. In classical compartment models, V DSS is the volume of distribution determined at steady state when the drug concentration in the tissue compartment is at equilibrium with the drug concentration in the plasma compartment (Fig. 10-9 Top)

49. Apparent Volume Distribution

50. In a physiological system involving a drug distributed to a given tissue from the plasma fluid (Fig. 10-9 bottom), the two-compartment model is not assumed, and drug distribution from the plasma to a tissue is equilibrated by perfusion with arterial blood and returned by venous blood. The model parameter V app is used to represent the apparent distribution volume in this model, which is different from V DSS used in the compartment model.

51. Apparent Volume Distribution V app is defined by the following equation : V app = D B C p The amount of drug in the body is given by the following equation : D B = V p C p + V t C t (2) where D B is the amount of drug in the body, V p is the plasma fluid volume, V t is the tissue volume, C p is the plasma drug concentration, and C t is the tissue drug concentration.

52. Apparent Volume Distribution For many protein-bound drugs, the ratio of D B /C p is not constant over time, and this ratio depends on the nature of dissociation of the protein–drug complex and how the free drug is distributed; the ratio is best determined at steady state. Protein binding to tissue has an apparent effect of increasing the apparent volume of distribution. Several V D terms were introduced in the classical compartment models. However, protein binding was not introduced in those models.

53. Apparent Volume Distribution Equation (2) describes the amount of drug in the body at any time point between a tissue and the plasma fluid. Instead of assuming the drug distributes to a hypothetical compartment, it was assumed that, after injection, the drug diffuses from the plasma to the extracellular fluid/water, where it further equilibrates with the given tissue. One or more tissue types may be added to the model if needed.

54. Apparent Volume Distribution If the drug penetrates inside the cell, distribution into the intracellular water may occur. If the volume of body fluid and the protein level are known, this information may be incorporated into the model. Such a model may be more compatible with the physiology and anatomy of the human body. Drugs such as penicillin, cephalosporin, valproic acid, and furosemide are polar compounds that stay mostly within the plasma and extracellular fluids and therefore have a relatively low V D.

55. Apparent Volume Distribution In contrast, drugs with lower distribution to the extracellular water are more extensively distributed inside the tissues and tend to have a large V D. An excessively high volume of distribution (greater than the body volume of 70 L) is due mostly to special tissue storage, tissue protein binding, carrier, or efflux system which removes drug from the plasma fluid.

56. Apparent Volume Distribution Digoxin, for example, is bound to myocardial membrane that has drug levels that are 60 and 130 times the serum drug level in adults and children, respectively. The high tissue binding is responsible for the large steady-state volume of distribution. The greater drug affinity also results in longer distribution half-life in spite of the heart's great vascular blood perfusion.

57. Protein Binding of Drugs Many drugs interact with plasma or tissue proteins or with other macromolecules, such as melanin and DNA, to form a drug– macromolecule complex. The formation of a drug protein complex is often named drug–protein binding. Drug–protein binding may be a reversible or an irreversible process. Irreversible drug–protein binding is usually a result of chemical activation of the drug, which then attaches strongly to the protein or macromolecule by covalent chemical bonding.

58. Protein Binding of Drugs Irreversible drug binding accounts for certain types of drug toxicity that may occur over a long time period. For example, the hepatotoxicity of high doses of acetaminophen is due to the formation of reactive metabolite intermediates that interact with liver proteins.

59. Protein Binding of Drugs Reversible drug–protein binding implies that the drug binds the protein with weaker chemical bonds, such as hydrogen bonds or van der Waals forces. Most drugs bind or complex with proteins by a reversible process. The amino acids that compose the protein chain have hydroxyl, carboxyl, or other sites available for reversible drug interactions.

60. Protein Binding of Drugs Reversible drug–protein binding is of major interest in pharmacokinetics. The protein-bound drug is a large complex that cannot easily traverse cell or possibly even capillary membranes and therefore has a restricted distribution (Fig. 10-11). Moreover, the protein-bound drug is usually pharmacologically inactive. In contrast, the free or unbound drug crosses cell membranes and is therapeutically active.

61. Diagram showing that bound drugs will not diffuse across membrane but free drug will diffuse freely between the plasma and extracellular water.